JP2001149087A - HETEROTROPHIC PRODUCTION OF MICROBIAL PRODUCT CONTAINING ω-3 HIGHLY UNSATURATED FATTY ACID AT HIGH CONCENTRATION - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of preparing an edible product for animal or the like by formulating a microorganism or the like selected from the group consisting of microorganisms belonging to the order Thraustochytriale, a ω-3 highly unsaturated fatty acid (ω-3 HUFA) extracted from the microorganism and these mixtures to a food. SOLUTION: This edible product is prepared by formulating the microorganism or the extracted ω-3 HUFA selected from the group consisting of microorganisms belonging to the order Thraustochytriale, the ω-3 HUFA extracted from the microorganism and these mixtures to an edible substance. As an actual example of the edible substance, a food for animal or a food for human being is illustrated and it is suitable to formulate an antioxidant to this edible product. Further, as an actual example of the above group, Thraustochytrium, Schizochytrium or the ω-3 HUFA extracted from Thraustochytrium is illustrated.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]Interaction with related applications  This application was filed on November 17, 1989,
-3 Microbial products containing high levels of polyunsaturated fatty acids
Are pendant together under the name "Rotropic manufacturing method"
Common Assignee U.S. Patent Application Serial No. 07 / 439,093
Is a continuation-in-part application. The original application is incorporated herein in its entirety.
Incorporated by reference in
Was filed on September 7, 1988, which was abandoned, and "ω-
3 Heterologous products of microbial products containing high levels of polyunsaturated fatty acids
US Patent Application No. 0, entitled "Tropic Manufacturing Method"
It is a continuation-in-part application of 7 / 241,410.

[0002]

The field of the invention is that of heterotrophic (heterotrophic) organisms and that they are suitable for human or animal consumption as food additives or for use in pharmaceutical and industrial products. The present invention relates to a method for culturing an organism for producing a lipid containing a high concentration of ω-3 polyunsaturated fatty acid (HUFA), which is suitable for the present invention.

In the description of the present invention, "ATCC number"
The Rockville, Maryland, United States
American Type Culture Collection (ATCC)
Means the accession number of the strain deposited at
TCC No. XXXXXX ”is the accession number XXXX of the strains deposited with the ATCC unless otherwise indicated.
X refers to the strain.

[0004]

BACKGROUND OF THE INVENTION Omega-3 polyunsaturated fatty acids have recently been recognized as important dietary compounds for preventing arteriosclerosis and coronary heart disease, reducing inflammatory conditions, and inhibiting tumor cell proliferation. And is of great industrial interest. These beneficial effects are that ω-3 polyunsaturated fatty acids cause competitive inhibition of compounds synthesized from ω-6 fatty acids and that beneficial compounds synthesized directly from ω-3 polyunsaturated fatty acids themselves. Produced by both (Simopoulos et al., 1986). ω-6 fatty acids are the main highly unsaturated fatty acids found in plants and animals. In recent years, the only commercially available source of omega-3 polyunsaturated fatty acids as commercial food is from certain fish oils, which can contain up to 20-30% of these fatty acids. The beneficial effects of these fatty acids can be obtained by eating fish several times a week or by taking concentrated fish oil daily. For this reason, large quantities of fish oil are processed and encapsulated each year for sale as edible supplements.

[0005] However, these fish oil supplements have several significant problems. First, they contain high levels of fat-soluble vitamins found naturally in fish oil. Upon ingestion, these vitamins accumulate and metabolize in human fat, rather than being excreted in urine. Large doses of these vitamins have safety implications in causing kidney damage and blindness, and some American medical communities have warned against using capsule supplements rather than real fish. ing. Second, fish oil contains up to 80% saturated and ω-6 fatty acids, both of which can have adverse health effects. In addition, fish oil has an intense fish taste and smell and cannot be added to processed foods as a food additive as it is without adversely affecting the taste of edible products. Furthermore, the isolation of pure ω-3 polyunsaturated fatty acids from this mixture involves an expensive process, so that pure products of these fatty acids are expensive ($ 200-1000 /
g) (Sigma Chemical Co., 1988; CalBio
chem Co., 1987).

[0006] The natural source of ω-3 polyunsaturated fatty acids in fish oil is algae. These polyunsaturated fatty acids are important components of the photosynthetic membrane. ω-3 polyunsaturated fatty acids accumulate in the food chain and are eventually incorporated into fish oil. Bacteria and yeasts cannot synthesize ω-3 polyunsaturated fatty acids, only a few molds are known to be able to produce trace amounts of ω-3 polyunsaturated fatty acids (Weete, 1
980; Wassef, 1977; Erwin, 1973).

[0007] Algae are grown in outdoor culture ponds to produce a variety of products containing ω-3 polyunsaturated fatty acids, including biomass, phototrophically. For example, U.S. Pat. No. 4,341,038 describes a photosynthetic process for producing oil from algae, and U.S. Pat. No. 4,615,83.
No. 9 describes a method for concentrating eicosapentaenoic acid (a type of ω-3 highly unsaturated fatty acid) photosynthetically produced by several types of green alga Chlorella. Phototrophic is the process by which cells use light as an energy source to build organic compounds from carbon dioxide and water through a photosynthetic process. Since sunlight is the driving hose (driving force) for this type of production system, a large surface area (large land) is required for the algae culture pond to be economically viable.
Need. The large scale, high cost and technical problems make it impossible to cover these systems economically. Another problem is that even a transparent cover can easily block a significant portion of sunlight. Therefore, such production systems are not aseptic and are difficult to maintain as a single culture. This is a particularly serious problem when it is necessary to manipulate or stress (eg, limit the amount of nitrogen) to produce the desired product in culture. Typically, it is during this period of stress that the cells readily produce products and do not proliferate, and the impurities readily enter the culture system. Thus, in many cases, the biomass produced is not desirable as a food additive for human consumption without a costly extraction process to recover the lipids. Moreover, the photosynthetic production of algae in open-air systems is very costly. This is because the culture system must be maintained at a low density (1-2 g / l) in order to avoid a decrease in the input light to the culture system. As a result, large volumes of water must be treated to recover small algae, and the algal cells are so small that costly harvesting processes must be used.

Mixed nutrition is an alternative method of production in which certain strains of algae perform photosynthesis using light as the required energy source, but additionally supply organic compounds into the system. Densification can be achieved by mixed nutrients and the culture can be maintained in a closed reactor for aseptic production. U.S. Pat. Nos. 3,444,647 and 3,316,674 describe mixed nutritional production of algae. However, due to the need to supply light to the culture medium, this type of production reactor is very expensive to build and operate, and the culture density is still very limited.

Another problem in algae culture for the production of ω-3 polyunsaturated fatty acids is that even though ω-3 polyunsaturated fatty acids constitute 20-40% of the total fatty acids of a strain, these The total fatty acid content of algae is generally very low, about 5 to 10% of the dry weight excluding ash. In order to increase the fatty acid content of the cells, there must be a nitrogen limitation period that stimulates lipid production. However, of all the strains described so far in the literature, of the 60 or more strains evaluated by the present inventors, all strains showed the percentage of ω-3 highly unsaturated fatty acids as a percentage of total fatty acids under nitrogen limitation. Resulted in a significant decrease (Erwin, 1973; Pohl & Zurheide, 1979).

From the viewpoint of economy and the use of ω-3 polyunsaturated fatty acids as food additives, it would be desirable to produce these fatty acids in a heotropic medium. Heterotropic (heterotrophic) refers to the capacity for sustained and continuous growth and cell division in the dark, with cellular carbon being obtained solely through the metabolism of organic compounds. Because light need not be supplied to the heterotrophic medium, cultures can be grown at very high density in a closed reactor. Heterotropic organisms are organisms that obtain energy and cellular carbon from organic substrates and can grow in the dark. Heterotropic conditions are those conditions under which heterotrophic organisms can grow regardless of the presence or absence of light. However, almost all algae are mainly phototrophic, and only a few heterotrophic algae are known. U.S. Patent No. 3,
Nos. 142,135 and 3,882,635 describe Chlorella, Spongiococcum,
And methods for heterotrophic production of proteins and pigments from algae such as Prototheca. However, these genera and others (for example, Scenedosmus), which are described as heterotrophically growing very well, do not produce ω-3 polyunsaturated fatty acids (Erwin, 1973). More or less ω
Very few heterotrophic algae known to produce -3 highly unsaturated fatty acids (eg, apochlorotic diatoms or apochlorotic diflagellates)
It is generally slow growing and produces small amounts of ω-3 polyunsaturated fatty acids, only a few percent of its dry weight excluding ash (Harring
ton and Holfz, 1968; Tornabene et al., 1974).

A few higher fungi are known to produce ω-3 highly unsaturated fatty acids, which make up only a small fraction of total fatty acids in cells (Er
win, 1973; Wassef, 1977; Weete, 1980). Thus, they are not considered good candidates for industrial production of ω-3 polyunsaturated fatty acids. For example, Yamada et al. (1987) recently reported the culture of several bacteria, Morfierella (isolated from soil), for the production of ω-6 fatty acids, arachidonic acid. When grown at low temperatures (5-24 ° C.), these bacteria produce small amounts of ω-3 eicosapentaenoic acid along with arachidonic acid.
However, the resulting eicosapentaenoic acid content is only 2.6% of the dry weight of the cells, and the low temperature required to promote the production of this fatty acid in these species depends on the production of the culture system. Resulting in a significant reduction in performance (and economy).

Some single cells of the order Thraustochytriales are also known to produce ω-3 highly unsaturated fatty acids (Ellenbogen, 1969; Wassef, 1977; Weete,
1980; Findlay et al., 1986), which make these difficult to culture. Sparrow (1960) is a frond of the Thraustochytriaceae
li) 's small size and simple nature make proliferation extremely difficult. Another reason for this difficulty is Eme
rson (1950)
976): "1) These fungi consist of very small fronds of only one or a few cells and are generally very slow growing and very sensitive to environmental changes; 2 ) They are generally saprophytic or parasitic plants with very specific nutritional and environmental needs;
3) In pure media, they generally show limited growth, vegetative growth that ends in a few generations. "
(Some prior references classify troost titride as a fungus, but the latest consensus states that it should be classified as an algae; see below). As a result, little attention has been paid to the numerous eyes of these microorganisms. The work done so far has been mainly based on taxonomic and ecological aspects. For example, it has been reported from a mixed nutritional point of view that some constituents of troost chitriale have a simple fatty acid distribution (Ellenbogen, 1969;
Findlay et al., 1986), there is no report of its total fatty acid or lipid content as a dry weight percent.
Unless data on total lipid content is available, the ability of an organism to be used for the production of any type of fatty acid cannot be assessed. For example, the ω-3 polyunsaturated fatty acid content in lipids of some marine macroalgae (sea algae) has been reported to be very high, up to 51% of total fatty acids (Pohl and Zurheide, 1979). . However,
The algae generally have a low lipid content and are 1% of the dry weight of the cells.
Only ~ 2% (Ryther, 1983). Thus, despite the reported high content of ω-3 polyunsaturated fatty acids in the fatty acids of macroalgae, the candidate organisms for the production of ω-3 polyunsaturated fatty acids are not Deemed eligible. Despite the intense search by the inventor, no simple approximate analysis (% protein, carbohydrates and lipids) of troost chitriale was found and a laboratory for its taxonomy, bioactivity or ecology. No attempt has been made to culture these species for purposes other than classical research. In addition, many species of these microorganisms have a simple pollen bait (pollen baitin).
g) method (eg, Gaertner, 196
8). The pollen biting method is very specific to members of the Troost chitriale, but does not select for any characteristics desired for large-scale culture of microorganisms.

As described above, up to the present invention, no heterotrophic organism suitable for cultivation that produces ω-3 highly unsaturated fatty acids at a practical level has been known, and such an organism is known as a natural environment. Some were considered extremely rare.

[0014]

The present invention relates to heterotrophic (heterotrophic) organisms and to their use in humans and animals as food additives or for use in pharmaceutical and industrial products. The object of the present invention is to provide an organism for effectively producing a lipid containing a high concentration of ω-3 polyunsaturated fatty acids (HUFA), which is suitable for the organism, a method for culturing the organism, and an edible product using the same. .

[0015]

SUMMARY OF THE INVENTION The present invention comprises high concentrations of ω-3 polyunsaturated fatty acids (HUFA), including microorganisms having a high concentration of fatty acids, most of which are ω-3 polyunsaturated fatty acids. Edible products. In addition, or
Separately, this edible product is ω-3HUF extracted from microorganisms
A. In particular, the microorganism is Thraustochytriale or Thraustochytrium or Schizochytrium. The microorganism or the extracted ω-3HUFA is added to the food material (edible substance) for addition. This food material may be either animal food or human food. The edible product of the present invention may increase the bioavailability of ω-3 HUFA contained therein by destroying microbial cells. The edible product may also be extruded. ω-3H
To prevent UFA degradation, the edible product may be packaged under non-oxidizing conditions or may further contain an antioxidant.

[0016] Another aspect of the present invention relates to a method of breeding an animal, comprising feeding the animal with Troost thytrium or ω-3HUFA extracted therefrom. Animals bred by the method of the present invention include marine organisms such as poultry, cattle, pigs and fish, shrimp and shellfish. ω-3HUFA
Is incorporated during the production of meat, eggs and other of these animals. ω-3HUFA is a whole cell microbial product, extracted ω-3H
Consumed in the form of a UFA product, or an animal or animal product incorporating ω-3HUFA. The higher the human consumption of ω-3HUFA produced according to the present invention, the more effective it is at preventing or treating cardiovascular diseases, inflammation and / or immunological diseases, and cancer.

[0017] Still another embodiment of the present invention provides a method comprising:
A method for producing ω-3 HUFA, comprising culturing Troost titriale with a nitrogen source that can be assimilated with an organic carbon source. Preferably, the nitrogen source that can be assimilated with the organic carbon source consists of ground grains. The method further comprises culturing Troost titriale consisting of Troost titorium, Schizochytrium or a mixture thereof under nutrient-restricted or nitrogen-restricted conditions for an effective time, preferably for about 6 to 24 hours, This involves then harvesting the Troost titriale during a nitrogen limitation period to increase the concentration of omega-3 HUFA in the microorganism. The method further comprises, during post-harvest processing, an antioxidant selected from the group consisting of BHT, BHA, TBHQ, ethoxycuin, β-carotene, vitamin E, and vitamin C to prevent degradation of ω-3HUFA. Including adding an oxidizing agent. The method further comprises stressing the microorganism at a low temperature during the culture, maintaining the medium at a high dissolved enzyme concentration during the culture, and an effective amount of phosphorus and a microbial growth factor to provide sustained growth of the microorganism. (Yeast extract or corn immersion liquid) to the culture medium. The method further comprises ATCC numbers 20888, 20889, 2089.
0, 20891, 20892 and mutants derived therefrom, including culturing unicellular microorganisms having similar properties. Ω-3 HUFA produced by these methods can then be isolated by fractional crystallization from lipids extracted from microorganisms. This method disrupts microbial cells, extracts the lipid mixture from the disrupted cells with a solvent, hydrolyzes the lipid mixture,
Remove unsaponifiable compounds and remove non-HU in the lipid mixture
Including cold crystallization of the FA.

Another embodiment of the present invention is a method for selecting a unicellular microorganism capable of heterotrophic growth and capable of producing ω-3HUFA and living in water. The method comprises selecting microorganisms of a size between about 1 μm and 25 μm from a small population of microorganisms collected from naturally occurring shallow seawater habitats, and comprising organic carbon, assimilable nitrogen, assimilable phosphorus, and microorganisms. Culturing the microorganism under heterotrophic conditions in a culture medium containing growth factors, and then selecting non-filamentous, transparent, white, orange or red filamentous colonies having a rough or textured surface. .

[0019]

DETAILED DESCRIPTION OF THE INVENTION As defined throughout this application, fatty acids are understood herein to mean aliphatic monocarboxylic acids. Lipids are phosphatides,
Steroids, alcohols, hydrocarbons, ketones,
And fats and oils, including glyceride esters of fatty acids with associated compounds.

A commonly used abbreviation system is used herein to describe the structure of fatty acids (eg, Weete, 1980). This system uses the letter "C" with a number indicating the number of carbons in the hydrocarbon chain, followed by a colon and a number indicating the number of double bonds. That is,
C20: 5 represents eicosapentaenoic acid. Fatty acids start numbering from the carbonyl carbon. The position of the double bond is represented by the Greek letter delta (Δ) with the number of carbon atoms of the double bond. In other words, C20: to Fu that 5ω-3Δ ^{5,8,11,14,17.} The “ω” designation is an abbreviated system for unsaturated fatty acids, where numbering is used from the carboxy terminal carbon. For convenience, w3 is used to symbolize "omega-3", especially when using numeric abbreviation nomenclature herein. ω-3 polyunsaturated fatty acids are understood to be polyethyleneic fatty acids in which the last ethylenic bond contains the terminal methyl group of the fatty acid and is then three carbons. Thus, the complete nomenclature of one ω-3 highly unsaturated fatty acid, eicosapentaenoic acid, is C20: 5w3Δ
^5,8,11,14,17 . For simplicity, the double bond position (Δ ^{5,8,1 1,14,17} ) may be omitted. Thus, eicosapentaenoic acid is indicated as C20: 5w3, and docosapentaenoic acid (C22: 5w3Δ ^{7,10,13,16,19} ) is C22: ^5w3.
w3 and docosapentaenoic acid (C22: 6w3Δ
^{4,7,10,13,16,19} ) is C22: 6w3. The designation “highly unsaturated fatty acids” refers to fatty acids having four or more double bonds. "Saturated fatty acid" means a fatty acid having 1 to 3 double bonds.

To facilitate the isolation of many microbial strains having the following combination of properties that are economically desirable for production of ω-3 polyunsaturated fatty acids, the inventors have developed collection and screening methods. : 1) capable of heterotrophic growth (proliferation); 2) high content of ω-3 highly unsaturated fatty acids; 3) single cells; 3) preferably low content of saturated and ω-6 highly unsaturated fatty acids; 5) Preferably, they are pigment-free, white or substantially colorless cells; 6) are preferably heat-resistant (can grow at a temperature of 30 ° C. or higher); and 7) are preferably broad-salt (broad-ranged) Can grow over low salinity, especially at low salinity).

Collection of a number of suitable heterotrophic strains,
Isolation and selection may involve the following methods. Suitable water samples and organisms can generally be collected, preferably from shallow saline habitats subject to wide temperature and salinity variability. These habitats include ocean tide pools, estuaries (rivers) and inland saline ponds, springs, playa and lakes. Specific examples of these collections include: 1) warm salt water springs, such as along the Colorado River at Glenwood Springs, Colorado, or along the western edge of the Stansbury Mountains, Utah; Playas, such as Goshen Playa, near Goshen, Utah; 3) Sea tide pools, such as in the Bardrox area of La Jolla, California; 4) Estuaries, such as the Chiajuana Estuary, in San Diego County, California. is there. Particular efforts are made to include some living plant matter and naturally occurring debris (degrading plant and animal matter) along with the water sample. The sample is then refrigerated until returning to the laboratory. Water sample 15 ~
After shaking for 30 seconds, for example, 1 to 10 ml is pipetted or partially poured into the filter unit to minimize the sampling error. The filter unit contains two types of filters. 1) a sterile Whatman # 4 filter (trademark, Whatman, Clifton, NJ) on top; and 2) a 1.0 μm pore size polycarbonate filter below the Whatman filter. The purpose of the first (upper) filter is to remove all particles of about 25 μm or larger in size, and generally only single cell type material permeates over a 1.0 μm polycarbonate filter Can. The first filter significantly reduces the number of mold colonies that subsequently grow in culturing polycarbonate filters at higher temperatures, increasing the chances of other colonies growing. Mold spores are very abundant in seashore and inland saltwater, and mold colonies can quickly cover the agar plate unless screened for. A 1.0 μm sized polycarbonate filter was chosen to allow most of the bacteria to pass through to the filtrate. The purpose of using a sandwich-type filter design is to select at least some of the cells in the range of about 1 μm to about 25 μm in diameter among unicellular organisms (in fermentation systems for large-scale production, Organisms that can potentially grow easily). Further growth of these unicellular organisms can be promoted by culturing polycarbonate filters on agar plates. Competition between organisms growing on the filters facilitates the isolation of competitive, robust strains of unicellular microorganisms. The unicellular aquatic microorganisms selected by the above-described method exhibit a range of cell sizes depending on the growth conditions and stages of the growth cycle. Most cells in culture have a diameter ranging from about 1 μm to about 25 μm. However,
Cells (thallus and sporangia) in culture can be found to have a larger diameter (up to about 60 μm), depending on the strain.

After filtration, the polycarbonate filter is placed on an agar plate containing saline. This culture medium is composed of organic carbon sources such as glucose, various starches, molasses, carbohydrates such as ground corn, assimilable organic or inorganic nitrogen sources such as nitrates, urea, ammonium salts, amino acids; enzyme extracts, vitamins and corn. It contains a microbial growth factor contained in one or more of the immersion liquids; an organic or inorganic assimilable phosphorus source; and a pH buffer such as bicarbonate. Microbial growth factors are unidentified compounds that promote heterotrophic growth of unicellular microorganisms, including fungi and algae. Agar plate is 25 in dark place
３５35 ° C. (preferably 30 ° C.)
After 2-4 days, a large number of colonies appear on the filter. It is not uncommon to recover 1 to 5 colonies of a target organism per plate. Yeast colonies can be distinguished either by color (often pink) or by their morphology. Yeast colonies are smooth, but the organism of interest forms colonies with a rough or textured surface. The organism of interest can see individual cells at the border of the colony through a microscopic microscope, but yeast cells are small and indistinguishable. Mold and higher fungal colonies can be distinguished from target organisms because they are filamentous, although the target organism is not filamentous. Clear or white colonies can be picked from the plate and re-streaked onto a new plate of similar medium composition. Most target organisms are clear or white, but some are orange or red due to the presence of the xanthophyll pigment, which is equally suitable for selection and restreak. A new plate can be cultured under similar conditions (preferably at 30 ° C.), and a single colony can be picked in a culture period of 2 to 4 days. Thereafter, a single colony can be picked and placed, for example, in 50 ml of liquid culture medium containing the same organic additives (except for agar) as in agar plates. These cultures can be cultured at 30 ° C. with blowing air, for example on a rotary shaking table (100-200 rpm) for 2-4 days. When the culture seems to have reached the highest density,
20-40 ml of the culture can then be harvested by centrifugation or other suitable method and stored, for example, frozen. The sample can then be analyzed by standard and well-known methods, including gas clostography, to identify the fatty acid content of the strain. Strains containing ω-3 highly unsaturated fatty acids can be identified in this way, and cultures of these strains can be maintained for further screening.

Promising strains were identified as 25 strains containing 50 ml of culture medium.
One can screen for temperature tolerance by inoculating the strain into a 0 ml shake flask. These cultures are then incubated for 2 days on a shaking table at any desired temperature range, most practically between 27-48 ° C, at 3 ° C intervals per culture. The amount of production can be quantified by the total amount of fatty acids produced per ml of culture medium. Total fatty acids can be determined by gas chromatography as described above. Similar methods can be used for screening for salt tolerance. For salinity tolerance, for most purposes, a salinity range that provides a conductivity of 5-40 mmho / cm is appropriate. Screening for the ability to use various carbon and nitrogen sources can also be performed using the methods outlined above. Here, the carbon source and the nitrogen source were evaluated at a concentration of 5 g / l. The carbon sources evaluated were glucose, starch, ground corn, potato starch, wheat starch and molasses. The nitrogen sources evaluated were nitrates, urea, ammonium, amino acids, protein hydrolysates, corn steep liquor (corn steep liquor), tryptone, peptone, or casein. Other sources of carbon and nitrogen can also be used, based on criteria that the user considers important,
Those skilled in the art can arbitrarily choose.

Unexpectedly, it has been found that a strain of a species from the genus Thrausochytrium produces fermentation of ground, unhydrolyzed grain directly to produce ω-3HUFA. Was. This method is advantageous over conventional fermentation methods because such cereals are generally cheap carbon and nitrogen sources. further,
Even if this method is carried out, such as the amount of ω-3HUFA,
Does not affect the beneficial properties of the algae.

The method using direct fermentation of cereals is not limited to corn, sorghum, rice, oats, rye,
And any kind of cereal, including millet.
However, it is preferred to use at least coarsely ground cereals, more preferably cereals ground to a flour-like consistency. This method further provides a low-cost source of carbon / nitrogen, as well as non-hydrolyzed corn syrup or stilla.
ge), the use of by-products in agricultural production and fermentation, and unnecessary products in fermenting corn to alcohol.

In another preferred method, ω-
It has been found that 3HUFA can be produced by Troost or Schizochytrium by fermentation of the above-mentioned hydrolyzed grains and unwanted products. Such cereals and waste products can be hydrolyzed by any method known to those skilled in the art, such as acid hydrolysis or enzymatic hydrolysis.
Another embodiment is a mixed hydrolysis treatment. In this method, the ground cereal is first partially hydrolyzed under mild acid conditions according to any mild acid treatment method known to those skilled in the art. Subsequently, the partially hydrolyzed crushed cereal was further
Hydrolysis by an enzymatic process according to any enzymatic method known to the person skilled in the art. In this preferred method, enzymes such as amylase, amyloglucosidase, α- or β-glucosidase or mixtures of these enzymes are used. The resulting hydrolysis products are then used in the present invention as carbon and nitrogen sources.

Using the collection and screening methods outlined above, single-cell fungal and algae strains of ω
-3 contains up to 32% of the ashless total cell dry weight (afdw) of the polyunsaturated fatty acid content and 15-48
One that grows in the temperature range of ° C. and grows in cultures with very low salinity can be isolated. Many very high ω-3 strains grow very slowly. A strain which is isolated by the above-described method, produces fast growth and produces ω-3 polyunsaturated fatty acids well, and has a high content thereof has an ω-3 polyunsaturated fatty acid content of about 15%.
% Up to about afdw. The growth of the strain according to the method of the invention is at any temperature at which satisfactory growth proceeds. For example, about 1
It can be achieved at 5C to 48C, preferably 25C to 36C.
The culture medium is generally adjusted by adding acid or buffer.
If H is not regulated, it will be more alkaline during fermentation. The strain grows in the pH range from 4.0 to 11.0, with the preferred range being about 5.5 to 8.5.

When the growth is carried out in a large volume container or tank, the antifreezing agent, dimethyl sulfoxide (DMS) is used.
It is preferred to prepare a vegetative transplant in a nutrient broth medium by transferring an aliquot from a culture or a gradient culture stored at -70 ° C with O) or glycerin into this broth culture. When a young, active nutritional implant is secured, it can be aseptically transferred to a larger production tank or fermenter. The culture medium from which the vegetative transplant is prepared may be the same or different from that used for large-scale production of cells, as long as the strain grows well.

The present inventors have determined that a single cell line of the order Thraustochytriales (containing ω-3 fatty acids) isolated and screened by the method described above was obtained from Emerson (1950) and S.
As foreseen by chneide (1976), it was found that after several generations it generally showed limited growth, stopping vegetative growth. However, the inventor has found that maintaining relatively high concentrations (eg, KH ₂ PO ₄ > 0.2 g / l) and / or using yeast extract or corn steep liquor (> 0.2 g / l) It has been found that by adding a nutritional supplement (a source of fungal growth factor), continuous culture growth of these unicellular fungi can be maintained. The ability to maintain growth for more than a few generations in liquid media is referred to herein as sustained growth. As a group, Troost Titolide strains appear to respond in a more favorable manner to phosphate addition than Schizochytrium strains, the latter appearing to require less phosphate . For nutraceuticals that supply fungal growth factors, corn steeped liquid
It can replace yeast extract and, in certain strains, has a better effect in achieving densification of the strain in the medium. Corn soak and yeast extract contain one or more growth factors necessary for the cells to grow.
Although growth factors are currently unknown, they are components of yeast extract and corn steep liquor, and either of these well-known nutritional supplements is sufficient. 50
% Carbon conversion efficiency (g of cell dry weight produced per 100 g of organic carbon added to the culture medium) can be easily achieved using this method.

By harvesting cells during the exponential phase of growth, a high protein, high ω-3 high unsaturated fatty acid microbial product can be obtained. If significantly higher lipids and products of ω-3 polyunsaturated fatty acids are desired, cultivation can be performed under nutrient restriction, preferably under nitrogen restriction, for a suitable period, preferably 6 to 24 hours. The culture may be transferred to a nitrogen-free medium, or the initial nitrogen content of the growth medium may be adjusted, preferably to provide a nitrogen deficiency late in the exponential phase. Short induction period, usually 6-24
As far as time is concerned, nitrogen restriction promotes total lipid production while maintaining high levels of ω-3 polyunsaturated fatty acids.
This stage of culture, when the culture distribution reaches its maximum cell density, is known as the stationary phase. The length of the induction period can be adjusted by increasing or decreasing the temperature, depending on the strain used. In addition, cells can be converted to high carbon /
It can be cultivated in a continuous form in a culture medium having a nitrogen ratio, and it is possible to continuously produce a cell biomass having a high lipid content (and a high ω-3 content). The single cell line of the heterotrophic microorganism isolated by the screening method described above contains three ω-3 polyunsaturated fatty acids, ie, C20: 5w3,
Containing high concentrations of C22: 5w3 and C22: 6w3,
20: 4w6 at very low concentrations. The ratio of these fatty acids can vary depending on the culture conditions and the strain used. The ratio of C20: 5w3 to C22: 6w3 can range from about 1: 1 to 1:30. C22: 5w3 and C
The ratio of 22: 6w3 is 1:12, so only trace amounts of C2
It can be up to 2: 5w3. In the strain without C22: 5w3, the ratio of C20: 5w3 to C22: 6w3 is about 1: 1.
From 1:10. Additional polyunsaturated fatty acids,
C22: 5w6 is produced by several strains, including all of the prior art strains (up to a ratio of C22: 6w3 fatty acid to 1: 4). However, C22: 5w6 fatty acids are
It is considered undesirable as a dietary fatty acid because it is converted back to 20: 4w6 fatty acids. The screening method described in the present invention, however,
ω-6 polyunsaturated fatty acids (C20: 4w6 or C2
2: 5w6) to facilitate the isolation of some strains that contain no (or less than 1%).

HUFA in microbial products, such as those produced by the present method, can be converted to less desirable unsaturated or saturated fatty acids when exposed to oxidizing conditions. However, saturation of ω-3 HUFA can be reduced or prevented by adding synthetic or naturally occurring antioxidants such as β-carotene, vitamin E and vitamin C to the microbial product.

A food or feed product by adding a synthetic antioxidant such as BHT, BHA, TBHQ or ethoxycuin or a natural antioxidant such as tocopherol to the product after processing the cells after harvesting. Can be added. The amount of antioxidant thus added is determined by the degree required by subsequent applications, such as, for example, product formulation, packaging, and desired shelf life.

[0034] The concentration of naturally occurring antioxidants may be determined by harvesting the fermentate at a stationary stage rather than during exponential growth, stressing the fermentation at low temperatures, and / or reducing the dissolved oxygen concentration of the culture medium. Can be adjusted by keeping it high. In addition, the concentration of naturally occurring antioxidants can be adjusted by changing culture conditions such as temperature, salinity, and nutrient concentrations. In addition, a biosynthetic precursor of vitamin E, such as L-tryptophan or L-phenylalanine, can be added to the fermentation medium for ingestion and subsequent conversion to vitamin E. Alternatively, compounds that prevent oxidation in combination with antioxidants (eg, ascorbic acid, citric acid, phosphoric acid) can be added to the fermentation for uptake by cells prior to harvesting. Furthermore, the concentration of trace amounts of metals, especially those that exist in more than one valence state and have suitable redox potentials (eg, copper, iron, manganese, cobalt, nickel), can increase the concentration of HUFA in processed cells. It should be kept to the minimum required for maximum growth, so as to minimize its potential to cause autoxidation.

Other products that can be extracted from the harvested cell biomass include proteins, carbohydrates, steroids, carotenoids, xanthophylls, and enzymes (eg, proteases). Strains that produce high levels of ω-6 fatty acids have also been isolated. Such a strain is
Useful for producing ω-6 fatty acids, which are useful raw materials for the chemical synthesis of prostaglandins and other eicosanoids. More than 25% of total fatty acids are ω-6
Strains producing as fatty acids have been isolated by the methods described herein.

The harvested biomass is dried (eg, spray-dried, tunnel-dried, vacuum-dried, or the like) and used as food or food supplement for any animal whose meat or product is consumed by humans. Can be used.
Similarly, the extracted ω-3 polyunsaturated fatty acids can be used as feed or food supplements. Alternatively, the harvested and washed biomass can be used directly (without drying) as a food supplement. To extend its shelf life, the wet biomass is acidified (approximately pH = 3.5-4.5) and / or sterilized or flash heated to inactivate the enzyme and then either under vacuum or Canned, bottled or packaged under a non-oxidizing atmosphere (eg, nitrogen or carbon dioxide).
The term "animal" means any organism belonging to the animal kingdom and includes without limitation any meat from which chicken, seafood, beef, pork, mutton is obtained. Seafood is
Obtained from, but not limited to, fish, shrimp, and shellfish. The term "product" encompasses any product other than meat obtained from such animals, including but not limited to eggs and other products. When fed to these animals, the ω-3HUFA in the harvested biomass or the extracted ω-3HUF
A is incorporated into the meat, eggs, or other products of these animals, increasing their ω-3 HUFA content.

In different animals, the desired ω-3 HUFA
It should be noted that there are various requirements to achieve the content. For example, in ruminants, the animal is ω-3HUFA
To protect this unsaturated fatty acid from being degraded or saturated by the rumen flora before digestion and absorption of it, certain encapsulation procedures for ω-3 HUFA are required. ω-3 HUFA can be “protected” by coating the oil or cells with proteins (eg, zeain) or other substances that are indigestible (or slow digesting) in the rumen. This allows fatty acids to pass through the rumen of the ruminant without damage. Before coating the cells or oil, the proteins and other "protecting" substances are dissolved in a solvent. Cells may be pelleted before coating with a protective agent. Animals with a high dietary conversion (eg, 4: 1-6: 1) have an omega-3 HUFA equivalent to animals with a low dietary conversion (2: 1-3: 1).
Higher concentration of ω-3HUF to achieve uptake of
A would be needed. The feeding regime can be further optimized in view of the animal's life span in which harvested biomass or extracted omega-3 HUFA must be provided to achieve the desired result.

In most dietary applications, the fat content of the harvested cells is about 25-50% afdw, and the other components are proteins and carbohydrates. The protein can be significantly beneficial as a nutrient for cells, as the several strains evaluated all have essential amino acids and are considered to be nutritionally balanced proteins.

In a preferred method, freshly harvested and washed cells containing ω-3 HUFA (harvested by belt filtration, rotary drum filtration, centrifugation, etc.) reduce the water content of the harvested cell paste to less than 40% moisture. To reduce, it can be mixed with any dry ground grain. For example, corn can be used, and such mixing allows the cell paste / corn mixture to be extruded directly using conventional extrusion methods. Extrusion temperatures and pressures can be adjusted to vary the degree of cell disruption (all from whole cells to 100% disrupted cells) in the extruded product. Extrusion of cells in this manner is advantageous because some of the antioxidants in the grain protect the fatty acids from oxidation, and the extruded matrix also helps protect oxygen from reaching the fatty acids easily.
It is believed that the -3HUFA content is not significantly reduced. Synthetic or natural antioxidants can also be added to the cell paste / cereal mixture before extrusion.
Direct extrusion of the cell paste / cereal mixture can greatly reduce drying time and cost, and the degree of cell disruption can control the bioavailability of ω-3 HUFA. The desired degree of cell disruption is
It depends on a variety of factors, including the permissible level of oxidation (probably increased oxidation as cell disruption proceeds) and the required degree of bioavailability by the animal consuming the extruded material.

The single cell strain isolated by the above-described method immediately aggregates and precipitates. This process depends on the medium
It can be promoted by adjusting H to 7.0 or less. By this method, the cell concentration can be easily increased 6 times within 1 to 2 minutes. Thus, this method can be used to pre-enrich cells before harvesting, or to enrich cells to very high densities before nitrogen limitation. Nitrogen limitation (to increase lipid production) can therefore be done in smaller reactors or can combine cells from several reactors into one reactor.

In recovering the microbial cells from the culture medium, many methods can be used. In a preferred recovery process, the cells produced by the method are recovered from the culture medium by conventional separation, such as filtration or centrifugation. The cells are then washed; frozen, lyophilized, or spray-dried; and then a non-oxidizing gas such as carbon dioxide or nitrogen before addition to the processed food or dietary product (to eliminate oxygen). May be stored under atmosphere.

Cellular lipids containing ω-3 polyunsaturated fatty acids can also be extracted by any suitable means, for example, by supercritical fluid extraction or extraction with a solvent such as chloroform, hexane, methylene chloride, methanol, and the like. It can also be extracted from microbial cells by evaporating under reduced pressure to obtain a concentrated lipid material. The ω-3 polyunsaturated fatty acids in this preparation further hydrolyze the lipids, and then are subjected to traditional methods such as urea addition or fractional distillation (Schlenk, 1954), column chromatography (Kates, 1986). Using or supercritical fluid fractionation (Hunter, 1987)
Can concentrate highly unsaturated components. Alternatively, cells can be disrupted or lysed, and lipids can be extracted with vegetable or other edible oils (Borowitzka and Borowitzka, 198).
8). The extracted oil can be refined by well-known processes routinely used for refining vegetable oils (eg, chemical or material refining). These refining processes remove impurities from the extracted oil before it is used or sold as an edible oil. The refining process consists of a series of processes: degumming, bleaching, filtering, deodorizing and polishing the extracted oil. After refining, the oil can be used directly as a feed or food additive to produce an omega-3 HUFA-rich product. Alternatively, the oil may be further processed and refined as described below, and then used for the above-mentioned and pharmaceutical uses.

In a preferred process, high purity ω-3
HUFA or a mixture of high purity HUFAs can be easily concentrated from the extracted oil. Harvested cells (fresh or dried) can be obtained by sonication, liquid shearing (eg, French press with a Menton-Gaulin homogenizer), bead milling, pressing under high pressure, freeze-thaw,
Disruption or permeation can be achieved by well-known techniques such as freeze pressing or enzymatic digestion of cell walls. Lipids from the disrupted cells are extracted using a solvent or solvent mixture such as hexane, chloroform, ether or methanol. Optional to remove the solvent (eg, by vacuum rotary evaporator where the solvent can be recovered and reused) and then convert the triglycerites to free fatty acids or esters of fatty acids, including base hydrolysis, acid hydrolysis or enzymatic hydrolysis The lipids are hydrolyzed using the well-known method. ω-3HUF
In order to minimize decomposition of A, the hydrolysis should be performed at the lowest possible temperature (eg, room temperature to 60 ° C.) and under nitrogen. After hydrolysis is complete, the unsaponifiable matter is removed by extraction with a solvent such as ether, hexane or chloroform. Thereafter, the remaining solution is acidified by the addition of an acid such as HCl and the free fatty acids are extracted into a solvent such as hexane, ether or chloroform. The solvent solution containing the free fatty acids is then added to the non-HU
Cool to a temperature that is low enough for the FA to crystallize but not too low for the HUFA to crystallize. Typically, the solution is cooled to a range from about -60 to about -74C. Crystallized fatty acids (saturated fatty acids and mono-, di- and triene-type fatty acids) can then be removed by filtration, centrifugation or sedimentation (while keeping the solution cool). HUF
A remains dissolved in the filtrate (or in the supernatant).
When the solvent in the filtrate (or supernatant) is removed, ω-3H
A fatty acid mixture having a purity of 90% or more of either UFA or HUFA having a carbon chain length of 20 or more is obtained. Refined ω
-3 polyunsaturated fatty acids are then added as food additives
It can be used for human nutritional supplements or for pharmaceutical use. For these uses, the purified fatty acids can be encapsulated or used directly. Antioxidants can be added to fatty acids to improve their stability.

The advantage of this method is that it removes saturated and monounsaturated fatty acids before cold crystallization by the urea complex method or other expensive methods such as supercritical carbon dioxide extraction and high performance liquid chromatography. That is, it is not necessary to perform a simple extraction method. The advantage is that complex oils containing as many as 20 fatty acids, such as fish oils (saturated, mono-, di-, tri- and polyene-type fatty acids are continuously contained, and as such, a series of overlapping crystallization temperatures) Rather than indicating)
An oil of a simple fatty acid composition such as that produced by troast titride (3-4 saturated or monounsaturated fatty acids and 3-4 HUFAs; two groups of fatty acids have distinct crystallization temperatures. (Separated) is used as a raw material for purification.

In a preferred method, ω-3HUFA
Rich oil can be produced by cultivating a strain of Thraustochytrium. After extracting oil from the cells by any of several well-known methods, the remaining extracted (excluding lipid) biomass, consisting primarily of proteins and carbohydrates, can be sterilized and returned to the fermentor. Among them, strains of Thraustochytrium can be recycled directly as nutrient sources (carbon and nitrogen sources). It is not necessary to pre-hydrolyze or pre-digest the cell biomass. Once digested by acid and / or enzymatic treatment, the extracted biomass of Thraustochytrium can be recycled in a similar manner.

As detailed above, whole cell biomass is used as a food additive to enhance the ω-3 polyunsaturated fatty acid content and nutritional value of processed foods for human consumption or animal feed. Can be used directly. When used as animal feed, ω-3 HUFA is incorporated into animal meat or other products. Complex lipids containing these fatty acids can also be extracted from whole cell products with solvents,
For pharmaceutical or nutritional purposes and for industrial use,
It can be used in a more concentrated form (eg, in an encapsulated form). Another aspect of the invention involves administering to a human ω-3 HUFA from the above sources for the treatment of various diseases.

As used herein, "treatment" refers to both providing a drug for treatment and prophylaxis. ω-3H
The dietary value of UFA is widely recognized in the literature, and human consumption of omega-3 HUFA produced according to the present invention is effective in treating cerebrovascular disease, inflammatory and / or immune diseases, and cancer. .

The present invention will be described in more detail by way of examples. Species meeting the above selection criteria have not heretofore been described in the prior art. By using these selection criteria, the inventor was able to screen about 10
More than 25 potentially promising strains were isolated from 00 samples. American Type Culture Collection
Of the approximately 20,500 strains of (ATCC), 10 were later identified as belonging to the same taxonomic group as the strains isolated by the present inventors. In that Collection,
Those strains that were still alive were obtained and used to compare to strains isolated and cultured in the manner described above. The results of this comparison are shown in Examples 5 and 6 below.

Since the filing of the original application of the present application, there has been a revision of the classification of Troost Titolide due to recent developments. Modern classification theorists combine them with algae. However, due to the taxonomic uncertainties that still remain, it would be best for the purposes of the present invention to consider these strains as troostochtolides (eye; Thraustochytriales, Thraustochytriaceae, genus; Thraustochytrium or Schizochytrium). The latest taxonomic changes are summarized below.

All strains of the unicellular microorganisms disclosed and claimed herein are members of the order Thraustochytrid. Thraustochytrids (Thysanoptera: Thraustochytrids) are marine eukaryotes with a strange taxonomic history. The question of the taxonomic position of Troost Titolide has recently been
Moss (1986), and Bahnweb and Jackle (1986), and Chamberlain and Moss (1988). For convenience, Troost Titolide was initially positioned by taxonologists along with other colorless flagellate fungi in algae (algae-like fungi). However, the name of the algae eventually dropped out of taxonomic status, and troost titride remained in the egg algae (Nippugella). Oocytes were initially hypothesized to be related to heterocompetes, and extensive ultrastructural and biochemical studies summarized by Barr (1983) supported this assumption. Egg algae were in fact accepted by Leedal (1974) and other algaeologists as being part of the heterocone algae. However, expediently due to their heterotrophic nature, egg algae and troost titride are studied more by mycologists (scholars studying fungi) than by algaeologists (scholars studying algae). Have been.

From another taxonomic point of view, among the innovative biologists, two general schools of ideas have emerged on how nucleated organisms arise. One theory proposes an exogenous source of membrane-bound organelles through a series of endosymbiosis (Margulis (1970); for example, mitochondria are derived from endosymbiosis of bacteria, chloroplasts are found in ovaries, and , Flakera is derived from spirochetes). Other theories suggest that membrane-associated organelles evolved gradually from non-membrane-associated systems of prokaryotic ancestors via the autogene process (Cavalier-Smith 1975). However, both groups of innovative biologists, however, have removed oocytes and troost titrides from the fungi and replaced them with brown algae of the brown plant kingdom (Calvalier-Smith 1981), or protoctistas (P.
rotoctista) Among all algae in the world (Margulis and Sag
an 1985).

With the development of electron microscopy, a study on the ultrastructure of the zoospores of two genus of troostrichtolide, troustyrium and schizochytrium (Perkins 1976; Kaz)
ama 1980; Barr 1981) provide good evidence that Troost titriaceae (Thysanoptera: Thraustochytriales) is quite distantly related to egg algae. Furthermore, 5S ribosome RN
More recent genetic data showing A sequence correspondence analysis (in the form of multivariate statistics) indicate that Troost chitriales are clearly a unique group in sexual organisms, completely isolated from fungi, Algae, brown algae, and egg algae, indicating the closest relationship (Mannella et al., 198
7). Recently, however, most taxonomies have agreed to remove troost titride from egg algae (Bartni
cki-Garcia, 1988).

In summary, Cavalier-Smith (1981, 19
Using the classification system of 83), troost titride is classified as a brown (chro-mophyta) algae in the brown plant kingdom, one of four plant kingdoms. This places them in a completely different kingdom from fungi that are all located in the kingdom Eufuguni. Therefore, the taxonomic position of troost titrides is summarized as follows. Genus: brown plant (chromophyta) phylum: heteroconta eyes: troost chitriale tribe: troost titriaceae (genus Thraustochytriales) genus: taxonomic within the genus and phyla, which is a higher class than troost titorium or Schizochytrium Despite its unclear position, Troost titride still exists as a distinctive distinctive group of its members, which can be classified in the order Troost titria.

Ω-3 polyunsaturated fatty acids are nutritionally important fatty acids for both humans and animals. Currently, the only source of these fatty acids that is commercially available is from fish oil. However, there are some significant problems when using fish oils as food or feed additives or supplements. First and most importantly, fish oils have an intense fishy odor and taste and cannot be added as such to processed foods without adversely affecting edible products. This means
The same is true for its many uses as an additive to animal food or feed. For example, experiments by the present inventors and others have shown that spawning hens immediately hate the diet when fed a diet rich in fish oil for more than a few days. Fish oil is very unstable and soon becomes putrid odor, which reduces the palatability and nutritional value of the food.

Second, fish oils generally have 20-30% ω-
Contains only 3HUFA. Desirable ω-3 HUFA content in marine juvenile and shrimp diets is 5 to 1% of its dry weight.
It can be as low as 0%. To constitute a suitable synthetic food containing 5-10% omega-3 HUFA, 15-30% requires a fish oil food. Such synthetic foods are not optimal for these young organisms in terms of taste, digestibility, or stability (Sargent
Et al., 1989). In terms of human nutrition, the other 70-80% of the fatty acids in fish oil are saturated fatty acids and ω-6 fatty acids, which can adversely affect human health. Including the process of isolating pure ω-3 fatty acids from fish oil is costly and the pure form of the resulting fatty acids can be very expensive ($ 200-1000 / g) and used as an additive in food and feed Too expensive (Si
gma Chemical, Co., 1988; CalBiochem Co., 1988).

Third, most baits currently used in the agricultural industry are cereal-based baits, which have relatively low ω-3 HUFA content. Recent surveys of marine products have revealed that fish and shrimps produced in aquaculture generally have only one-third to one-half the ω-3 HUFA content of wild-caught fish and shrimps. (Pigott, 1989). Aquaculture organisms are often praised for their mildness and lack of fishy odor, but increasing the fish oil content of their food is not as effective as a fish-tasting product. Absent.

As a result of the above problem, ω-3HUF
Developing another (non-fish-derived) source of A exists as a significant challenge.

The microbial products of the present invention can be used as food or feed supplements and provide a better source of ω-3 polyunsaturated fatty acids with significant advantages over conventional sources. Poultry raised on food supplemented with this microbial product,
Incorporates ω-3 highly unsaturated fatty acids into body tissues and eggs. This egg has no fishy odor and no fishy taste. Also, there is no change in the color of the yolk. Poultry does not stop eating its supplemental diet as it does on a fish oil supplemented diet.
The bait to which the microbial product of the present invention is added has a normal shelf life and does not emit putrid odor even when left at room temperature for several days. In accordance with the present invention, bred poultry eggs and meat are useful in human nutrition as a source of ω-3 highly unsaturated fatty acids, while having a low ω-6 fatty acid content and no fishy odor.

The microbial products of the present invention are also valuable as sources of ω-3 highly unsaturated fatty acids for fish, shrimp, and other products produced in aquaculture. The product can be added directly to the diet as a supplement, or it can be fed to salted shrimp or other live prey for consumption by aquaculture products. By using such supplements, high ω-3, such as wild fish, with the additional health benefits of reduced ω-6 fatty acid content, while retaining the taste benefits provided by aquaculture. Improved products with a high unsaturated fatty acid content can be marketed by fish and shrimp farmers.

[0060]

Embodiment 1Collection and screening  Collect 150ml water sample from shallow, inland saltwater pond
And stored in sterile polyethylene bottles. With water sample
With some living plant material and naturally occurring debris (degraded
(Plants and animals that are growing).
Strengthened. Samples were kept on ice until returning to the laboratory. Experiment
In the chamber, shake the water sample for 15 to 30 seconds, and
1 to the filter unit having a filter of Ip
A 10 ml sample was pipetted and injected. Two files
Luther, 1) At the top, about 25μm in diameter, 47mm in diameter
Sterile Whatman # 4 filter and 2) Whatman
Under the filter, a hole with a hole of about 1.0 μm and a diameter of 47 mm
It is a carbonate filter. Nominal hole of filter
By slightly changing the diameter in this way, polycarbonate
The size of the cells collected on the
μm to about 25 μm.

The Whatman filter was removed and discarded. Solid F in Petri dish with polycarbonate filter
-1 was placed on a culture medium. The culture medium contained the following per liter: 600 ml of seawater (we can use artificial seawater), 400 ml of distilled water, 10 g of agar, 1 g of glucose, 1 g of protein hydrolyzate, 0.2 g of yeast extract.
g, 0.1 MKH ₂ PO ₄ 2 ml, vitamin solution (A-vit
s), 1 m (containing 100 ml / l thiamine, 0.5 mg / l biotin, and 0.5 mg / l cyanocobalamin)
1, 5 ml of trace metal mixture (PII metal, 6.0 g of Na ₂ EDTA, 0.29 of FeCl ₃ .6H ₂ O per liter)
_{g, H 3 BO 3 6.84g,} MnCl 2 · 4H 2 O 0.86g,
0.06 g of ZnCl ₂ , 0.29 g of CoCl ₂ .6H ₂ O, (N
_{iSO 4 · H 2 O 0.052g,} CuSO 4 · 5H 2 O 0.00
2 g, and 0.005 g of Na ₂ Mo ₄ .2H ₂ O, and 50
0 mg of streptomycin sulfate and penicillin G. The agar plate was incubated at 30 ° C. in the dark. After 2 to 4 days, countless colonies appeared on the filter. Colonies of unicellular fungi (excluding yeast) were removed from the plates and restreaked on fresh medium plates of similar composition. Particular care was taken to pick out all colonies consisting of colorless or white cells. New plates were incubated at 30 ° C and single colonies were picked after an incubation period of 2-4 days.
Single colonies were picked and then placed in 50 ml broth containing the same organic enhancers as in the agar plates. Rotating and shaking the culture medium (100-200 rpm)
And incubated at 30 ° C. for 2-4 days. When the culture appears to have reached maximum density,
Cultures were harvested, centrifuged and frozen. The sample was then analyzed by standard well-known gas chromatography methods (eg, Lepage and Rey, 1984) to identify the fatty acid content of the strain. Thus, ω-
Strains containing the three highly unsaturated fatty acids were identified and cultures of these strains were further screened.

Using the collection and screening procedures described above, more than 150 strains of unicellular fungi were isolated. These strains have an ω-3 polyunsaturated fatty acid content of up to 32% of the total cell dry weight, excluding ash, and grow at temperatures ranging from 15 to 48 ° C. Less than 1% undesired C20: 4w6 (as% of total fatty acids) and C22: 5w
Strains containing 6 highly unsaturated fatty acids can also be isolated.
Strains of these fungi can be repeatedly isolated from the same location using the methods described above. A few newly isolated strains have very similar fatty acid profiles. It is currently undeniable that some may be double isolates of the same strain. Further screening for other desirable characteristics, such as salinity tolerance or the ability to use various carbon and nitrogen sources, can be performed subsequently using similar methods.

[0063]

Embodiment 2Maintaining unrestricted cell growth: phosphorus  A strain isolated by the method of Example 1, Troostylium sp.
U42-2 (ATCC No. 20891) cells were transformed into solid F-
Remove from the medium and add 50 ml of modified FFM medium (Fu
ller et al., 1964). This culture medium contains seawater 10
00 ml; glucose 1.0 g; gelatin hydrolyzate 1.
0 g; liver extract 0.01 g; yeast extract 0.1 g; PI
I metals 5 ml; 1 ml of B-vitamin solution (Goldstei
n et al., 1969); and 1 ml of antibiotic solution (25 g / l)
Streptomycin sulfate and penicillin G).
1.0 ml of vitamin mix (pH 7.2) is thiamine
Hydrochloride 200 μg; biotin 0.5 μg; cyanocoba
Min 0.05 μg; Nicotinic acid 100 μg; Pantothene
Calcium acid 100 μg; riboflavin 5.0 μg;
Lidoxin hydrochloride 40.0 μg; pyridoxamine dihydrochloride
Salt 20.0 μg; p-aminobenzoic acid 10 μg; chlorin
Hydrochloride 500 μg; inositol 1.0 mg; thymine 0.
8 mg; orotic acid 0.26 mg; folinic acid 0.2 μ
g and 2.5 μg of folic acid. 50ml of this culture medium
250 ml Erlenmeyer flask with 27 ° C orbital shake
It was placed on a container (200 rpm) for 2-4 days. at this point
The culture has reached its highest density. 1 ml of this culture
And add one extra treatment agent per liter
Transferred to a new flask containing the modified FFM medium:
 1) 1 ml of B-vitamin mix; 2) 1 ml of A-vita
Min solution; 3) 5 ml of PII metals; 4) 2 ml of 0.
1MKH_TwoPO_Two(-28 mg); 5) Treatment of 2, 3, and 4
6) 480 mg of KH_TwoPO_Four.
1 ml of culture, also modified without extra additives
Inoculate flasks of FFM medium and control
Roll. Rotating the culture with a rotary shaker (200 rpm)
And incubated at 27 ° C. for 48 hours. Then,
The cells are harvested by centrifugation and the fatty acids are
Quantified by luffy. The results are shown in FIG.
In FIG. 1, the yield is expressed as the ratio to the control.
The relative yield of the control is therefore 1.
0. The processing of 1 to 6 is as follows: 1) 2 × B Vitamins
2) Concentration of 2 × A vitamins; 3) 2 × fine
4) 2 x (B vitamins + phosphate +
5) 2x concentration of phosphoric acid; and
6) 24 mg of phosphate per 50 ml (0.1 per liter)
48g). 0.48 g of KH per liter_TwoO_FourAdd
Treatment alone results in enhanced growth and significantly increases
Fatty acid yield.

[0064]

[Table 1]

[0065]

Embodiment 3Maintaining unrestricted growth: PO4 and yeast extract  Schizuchytrium aggrega
tum) (ATCC No. 28209) on a solid F-1 medium.
Remove from nutrient medium and inoculate 50ml FFM medium
Was. The culture is placed on a 27 ° C. rotary shaker (200 rpm).
placed. After 3-4 days, 1 ml of this culture was
The following treatment was transferred to 50 ml: 1) FFM medium (con
And 2) KH_TwoPO_Four 250mg / l
And an FFM culture medium to which 250 mg / l of yeast extract was added. This
These cultures are placed on a rotary shaker (200 rpm) at 27 ° C.
For 48 hours. Harvest cells and quantify cell yield
Was. In treatment 1, fine weight on a dry weight basis excluding ash
The final concentration of vesicles was 616 mg / l. In process 2,
The final concentration of the vesicles is 1675 mg / l,
_FourIncrease in yeast and yeast extract concentration has a promoting effect.
Are shown.

[0066]

Embodiment 4Maintaining unrestricted growth: Yeast extract with corn
Substitute with immersion liquid  Schizochytrium sp.S31 (ATCC No. 20888)
Of cells from the solid F-1 culture medium and 50 ml of M-
Five culture media were added. This medium is (per liter
), 25 g of NaCl; MgSO_Four・ 7H_TwoO 5g; KCl
1 g; CaCl_Two 200mg; Glucose 5g; Glutamate
 5g; KH_TwoPO_Four 1 g; PII metals 5 ml; A-Vita
1 ml of Min solution; and 1 ml of antibiotic solution
You. Adjust the pH of the solution to 7.0 and filter-sterilize the solution
did. Corn immersion liquid (4 g / 40 ml; pH 7.0) and yeast
Prepare sterile solution of mother extract (1g / 40ml; pH 7.0)
did. Add the following amount of yeast to one set of M-5 culture flasks.
The extract solution was added: 1) 2 ml; 2) 1.5 ml; 3) 1
4) 0.5 ml; and 5) 0.25 ml. M-5 culture medium
The other set of flasks contains the following amounts of yeast extract and
And corn immersion liquid were added: 1) 2 ml yeast extract; 2)
1.5 ml of yeast extract and 0.5 ml of corn soak; 3)
1.0 ml yeast extract and 1.0 ml corn immersion liquid; 4)
0.5 ml yeast extract and 1.5 ml corn steep solution; and
 5) 2 ml of cone immersion liquid. 1m of culture in F-1 medium
1 was used to inoculate each flask. 27 ° C
Was placed on a rotary shaker for 48 hours. Centrifuge cells
Cell yield (dry weight excluding ash)
As). Table 2 shows the results. This result is
When the yeast extract is added to 0.8 g / l of the culture vessel, the cell yield
Is increasing. However, corn soak
The addition of the pickling solution is more effective,
The result was twice the theoretical yield. Cone immersion liquid
This is cheaper than yeast extract,
Is very advantageous for economical production of cells.

[0067]

[Table 2]

[0068]

Embodiment 5ATCC shares (formerly known strains)
In comparison, the highly unsaturated fat of the strain isolated by the method of Example 1
High fatty acid content.  151 selected according to the method described in Example 1
The battery of newly isolated strains in late exponential phase
Sampled by gas-liquid chromatography.
The unsaturated fatty acid content was quantitatively analyzed. All strains
Means that the cells give the highest yield,
Grow in any of the somatic FFM media. further,
Five previously isolated troustyrium or thirst
Zochithotrium species were purchased from American Type Culture Collec
Everything that can be obtained from this institution and alive from this institution
Representative of all strains. T. aureum (ATCC No. 2
8210), T. aureum (ATCC No. 3430)
4), T. roseum (ATCC No. 28210), T. strike
Lightum (ATCC No. 34473) and S. Agre
Gatum (ATCC No. 28209). These strains
All show growth in conventional media and are generally M5 media
And in the culture medium of the present invention containing FFM culture medium.
(Example 2). Fat of each known strain
Acid production leads to improved growth of the strain in the culture medium of the present invention.
And measured as described above.

The peak of the fatty acid was identified using a pure compound having a known structure. Quantification of the total fatty acids as% by weight was performed by integration of the chromatographic peaks. The compounds identified were palmitic acid (C16: 0), C20: 4w6
And C22: 1 (peaks were not separated in the system used), C20: 5w3, C22: 5w6, C22: 5w
3 and C22: 6w3. Residues, which are usually low molecular weight fatty acids, were included in the "Other fatty acids" category. Total ω-3 fatty acids are 20: 5w3, 22: 5
Calculated as the sum of w3 and 22: 6w3. Total ω-6 fatty acids were calculated as the sum of the 20: 4/22: 1 peak and 22: 5w6 peak.

The results are shown in Tables 3 and 4 and in FIGS. From Table 3, a large number of strains can be isolated by the method of the invention, and a large number of strains outperform previously known strains in some important criteria. For example, 10
2 strains have at least 7.8% by weight of total fatty acids of C20: 5
w3, and the percentage of this fatty acid is higher than any previously known strain. Strains 23B (ATCC No. 20892) and 12
B (ATCC No. 20890) is an example of such a strain. Thirty strains of the present invention produced at least 68% by weight of total fatty acids as omega-3 fatty acids, more than any previously known strain. Strain 23B (ATCC number 20892) is an example of such a strain. Seventy-six strains of the present invention produce less than 10% by weight of total fatty acids as omega-6 fatty acids, which are considered undesirable as human food components, which is lower than any previously known strain. Strains 23B (ATCC No. 20892) and 12B (ATCC No. 20890)
Is an example of such a strain. In addition, there are 35 strains of the present invention that produce at least 25% by weight of total fatty acids as omega-6 fatty acids, which is greater than any previously known strain. Although such strains are useless for food applications, they are useful as feed for chemical synthesis of eicosanoids from ω-6 fatty acids.

Further, the data indicate that many strains of the present invention produce a high proportion of total ω-3 fatty acids as C22: 6w3. In Table 4, the 48 strains shown in Table 2 were compared with the conventionally known strains in comparison with C20: 5w3, C2
Each of 2: 5w3 and C22: 6w3 is indicated by weight% of the total ω-3 content. Fifteen strains had at least 94% of the total omega-3 fatty acids in C2
2: 6w3. Strain S8 (ATCC No. 2
0889) was an example of such a strain. Eighteen strains had at least 28% by weight of total ω-3 fatty acids as C20: 5w3, more than any previously known strain. Strain 12B (ATCC number 20890) was an example of such a strain.

FIG. 2 shows the method of Example 1 with more than 67% ω-3 fatty acids (as% of total fatty acids) and less than 10.6% ω-6 fatty acids (as% of total fatty acids). A series of isolated strains is shown. All previously known strains have less than 67% omega-3 fatty acids (as% of total fatty acids)
And greater than 10.6% ω-6 (as% of total fatty acids).

FIG. 3 shows that more than 67% of ω-3 fatty acids (as% of total fatty acids) and more than 7.5% of C20: 5w3
1 shows a series of strains isolated by the method of Example 1 having (as% of total fatty acids). All previously known strains have less than 67% omega-3 fatty acids (as% of total fatty acids)
And less than 7.8% C20: 5w3 (as% of total fatty acids).

[0074]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

[0075]

[Table 8]

[Table 9]

[Table 10]

[0076]

Embodiment 6Compared to ATCC strains (known strains),
Strain isolated by the method of Example 1 shows improved growth rate
thing.  Schizochytrium sp.S31 (ATCC No. 2088)
8), Schizotylium sp. S8 (ATCC No. 2088)
9), Troostillium sp. S42, Troost chili
U. sp. U42-2, Troost Titrium sp. S42
And U30 (all isolated by the method of Example 1) and Trous
Totitrium and aureum (ATCC No. 28211)
And Schizochytrium, Aggregatam (ATCC No. 28
209) (a conventionally known strain) was taken from a solid F-1 culture medium,
It was placed in 50 ml of M-5 culture medium. This medium is (1 liter
Per g of yeast extract, 1 g of yeast extract; 25 g of NaCl;_Four
・ 7H_TwoO5g; KCl 1g; CaCl_Two 200 mg; guru
Course 5g; Glutamine 5g; KH_TwoPO_Four 1 g; PII
Metals 5 ml; A-vitamin solution 1 ml; antibiotics 1 m
l. The pH of the solution was adjusted to 7.0 and the solution was
Filtered and sterilized. Orbital shaker (200rpm,
27 ° C.) for 3 days, then 1-2 m of each culture
1 to another flask in M-5 medium and shake for 2 days
Placed on a bowl. The culture (1-2 ml) is then cultured in M-5.
Transfer the other flask containing the group and place it on a shaker for one day.
Was. This method ensures that all cultures are in the exponential phase of growth.
It was certain that there was. These late cultures are
Then, for each strain, add two M-5 culture media.
Used to inoculate a 250 ml flask. afterwards,
Place these flasks on a shaker at 25 ° C and 30 ° C.
And the change in their optical density is measured by Beckman DB-G spectroscopy.
Monitoring was performed with a photometer (660 nm, 1 cm optical path length). Optics
Density readings are 0, 6, 10, 14, 17.25, 2
Performed each time at 0.25 and 22.75. Then the index
The growth rate (doubling / day) was determined by the method of Sorokin (1973).
Thus, it was calculated from the optical density data. The results are shown in Table 5.
(Normalized for growth of strain U30 at 25 ° C.)
FIG. 5). From the data, isolated by the method of Example 1.
Strains have a sustained growth compared to previously known ATCC strains.
Even at the optimal phosphoric acid concentration essential for
Have a much higher growth rate at both ° C
Is clear. Trousers isolated from cold Antarctic waters
It was found that the strains of the order Candida do not grow at 30 ° C.
You.

[0077]

[Table 11]

[0078]

Embodiment 7Compared to ATCC stock (prior art stock),
The strain isolated by the method of Example 1 has better production characteristics (growth)
And lipid-induced).  Schizochytrium sp.S31 (ATCC No. 2088)
8), Schizochytrium sp. S8 (ATCC No. 2088)
9) (both isolated by the method of Example 1), and Trous
Totrium Thorium (ATCC No. 2821)
1), Schizochytrium aggregatam (ATCC No. 28)
209) The cells of (prior art strain) were transformed into solid F-1 medium.
And place in 50 ml of M-5 medium (see Example 5)
Was. The pH of the container was adjusted to 7.0, and the solution was sterilized by filtration.
Increased for 3 days on an orbital shaker (200 rpm, 27 ° C)
After inoculation, 1-2 ml of each culture was added to M-5 medium.
Transfer the other flask and place it on a shaker for 2 days
Was. Then, each of these cultures was dried to remove the ash.
Quickly measure dry weight and add 3.29 mg of each culture to 50 ml
Into two 250 ml Erlenmeyer flasks containing the M-5 culture medium.
Put in pets. Rotate these flasks on a rotary shaker (2
00 rpm, 27 ° C.). After 24 hours, each culture
, Centrifuge 20 ml each and discard the supernatant.
2 containing 50 ml of M-5 medium without nitrogen (nitrogen source)
The cells were transferred to a 50 ml Erlenmeyer flask. Shake the flask
Put it on the bowl again, and after another 12 hours,
To determine the dry weight, excluding ash,
The fatty acid content was determined by the method of e and Roy (1984).
The results (yield of conventionally known strain, ATCC No. 28211)
FIG. From these results, the example
The strain isolated by the method 1 is different from the prior art ATCC strain.
Also a combination of exponential growth and nitrogen restriction (for lipid induction)
Under the same time, with the dry weight excluding ash,
It is found to produce twice as much. Further, the strain of the present invention
The yield of total fatty acids and ω-3 fatty acids was improved,
31 (ATCC No. 20888), the conventional AT
It produced 3-4 times more ω-3 fatty acids than the CC strain.

[0079]

Embodiment 8Improved by the strain isolated by the method of Example 1
Salt tolerance and fatty acid production  Schizochytrium sp.S31 (ATCC No. 20888)
And Troost Titrium sp.U42-2 (ATCC number
20891) (both are isolated by the method of Example 1 and
Leaning) and S. aggregatam (ATCC number
28209) and T. aureum (ATCC No. 2821)
0) (obtained from American type Culture Collection)
Of the four strains of Thraustochytrium in solid F-1 culture medium
And on a rotary shaker (200 rpm) at 27 ° C for 3 ~
Incubated for 4 days. A range of different salinities
Of the culture medium was treated with M medium salts (NaCl, 25 g / l; M
gSO_Four・ 7H_TwoO, 5 g / l; KCl, 1 g / l; CaC
l_Two, 200 mg / l) and adjust as follows:
Was. 1) 100% (w / v M culture base salts; 2) 80%
(V / v) M culture salts, 20% (v / v) distilled water; 3)
60% (v / v) M culture salts, 40% (v / v) distillation
Water; 4) 40% (v / v) M culture salt, 60% (v / v)
v) distilled water; 5) 20% (v / v) M culture salt, 80
% (V / v) distilled water; 6) 15% (v / v) M culture salt
, 85% (v / v) distilled water; 7) 10% (v / v) M medium
Nutrient salts, 90% (v / v) distilled water; 8) 7% (v / v)
M medium, 93% (v / v) distilled water; 9) 3% (v / v)
v) M culture salts, 97% (v / v) distilled water; 10) 1.
5% (v / v) M culture salt, 98.5% (v / v) distillation
water). The following nutrients were added to the processed material (1 liter
R): Glucose 5g; Glutamate 5g; Yeast extract
1 g; (NH_Four)_TwoSO_Four 200 mg; NaHCO_Three 200m
g; KH_TwoPO _Four 1 g / l; PII metals 5 ml; A-Vita
1 ml of a min solution; and 2 ml of an antibiotic solution. These processes
50 ml of each substance grown in F-1 medium
1 ml of cells was inoculated. These cultures were orbital shaked.
Place on a centrifuge (200 rpm) and maintain at 27 ° C. for 48 hours.
Was. Cells are harvested by centrifugation and fatty acids are
Quantification was by chromatography. FIG. 7 shows the results. Implementation
Thraustochytrium sp. U42 isolated by the method of Example 1
-2 (ATCC No. 20891) is T. aureum (A
TCC No. 28210), almost twice the fatty acids produced,
The production of S. aggregatam (ATCC No. 28209)
Can produce more than 8 times the amount of fat cells produced
You. In addition, U42-2 is at the upper end of the tested salinity range.
Would have a wider salt tolerance. Yaha
Schizochytrium sp. S31 isolated by the method of Example 1
(ATCC No. 20888) is a conventionally known ATCC strain.
Fatty acid yield (ATCC strain known in the art)
2.5 to 10 times the yield of) and a very wide range of salt concentrations
Degree tolerance was shown. Besides, Schizochytrium sp.S3
1 (ATCC No. 20888) at very low salinity
Grow best. This property is important for saltwater metal reactors.
For both the corrosive action of the seawater and the problems associated with the disposal of salt water
Also has important economic advantages when considering factory production.
Sprinkle.

[0080]

Embodiment 9Culture / low salinity  50 of M / 10-5 medium in a 250 ml Erlenmeyer flask
ml of Schizochytrium sp. taken from the agar slant culture.
S31 (ATCC No. 20888) colony
Was. This M / 10-5 culture medium has 1000 m of deionized water.
1, NaCl 2.5 g, MgSO_Four・ 7H_TwoO 0.5g, KC
l 0.1 g, CaCl_Two 0.02 g, KH_TwoPO_Four 1.0g,
Yeast extract 1.0 g, glucose 5.0 g, glutamic acid
5.0 g, NaHCO_Three 0.2 g, PII trace metals 5 m
l, vitamin mix 2ml, and antibiotic mix
Contains 2 ml. The culture is a rotary shaker at 30 ° C.
(200 rpm). 2 days later, culture
Had a moderate density and were actively growing. This activity
Add 20 ml of the growing culture to glucose and glue.
Tamate concentration of 40 g / liter (M / 10-40 culture
2 liters containing 1700 ml of the same culture medium except that
Used to inoculate torr fermenters. Fermenter at 30 ℃
And aeration at 1vol / vol / min, 300r
Stirred at pm. After 48 hours, the cell concentration in the fermentor was 2
It became 1.7 g / l. Harvest cells by centrifugation and freeze
And stored under nitrogen.

The total fatty acid content and the ω-3 fatty acid content were measured by gas chromatography. The total fatty acid content of the final product was 29.0% by dry weight, excluding ash. Ω-3 highly unsaturated fatty acid content of microbial products (C20:
5w3, C22: 5w3 and C22: 6w3) were 15.6% of the dry weight excluding ash. The ash content of the sample was 7.0%.

[0082]

Example 10 By gas chromatography analysis of the growth and fatty acid production of the various strains described in Example 5,
The difference in fatty acid distribution became apparent. The strains of the present invention synthesized fewer types of fatty acids than previously available strains. A small fatty acid dispersion is advantageous because there are few impurities to be separated. For the purpose of food supplements, a reduced variety of fatty acids is advantageous, as it reduces the possibility of ingesting unwanted fatty acids. Table 6
Indicates the number of types of polyunsaturated fatty acids present at concentrations exceeding 1% by weight of total fatty acids for the conventionally known strains and the various strains of the present invention indicated by the ATCC symbol.

[0083]

[Table 12]

[0084]

Embodiment 11Collection  50 ml of the M5 medium in a 250 ml Erlenmeyer flask was
Schizochytrium sp. S31 (AT
(TC No. 20888). M
/ 5 medium is 1000 ml of deionized water, NaCl 2
5.0g, MgSO_Four・ 7H_TwoO 5.0 g, KCl 1.0 g, C
aCl_Two 0.2 g, KH_TwoPO_Four 1.0 g, yeast extract 1.0
g, glucose 5.0 g, glutamic acid 5.0 g, NaHC
O_Three 0.2g, PII trace metals 5ml, vitaminmick
And 2 ml of antibiotic mix. Culture
Nutrition is ink on a rotary shaker (200 rpm) at 30 ° C.
Was added. After two days, the culture is at medium density.
And actively proliferated. This actively growing culture
20 ml of the product and a concentration of glucose and glutamate of 40
g / liter (M / 20 medium)
1 liter fermenter containing 1000 ml of the same culture medium
Used for inoculation. Fermenter at pH 7.4 at 30 ° C
Keep aeration at 1vol / min and 400rpm
And stirred. After 48 hours, the cell concentration in the fermentor was 18.
It was 5 g / l. Fermentation tank aeration and agitation
stopped. Within two to four minutes, the cells clump together and form the bottom of the fermenter.
Settle to 250 ml. The concentrated zone of this cell is 7
It had a cell concentration of 2 g / l. This cell zone,
Siphon out of fermenter and (1) nitrogen control
Transfer to another reactor for a limited time (for example, some
Together with the highly concentrated products of the fermenter)
Or (2) harvest directly by centrifugation or filtration.
Can be. By pre-concentrating the cells in this way,
60-80% less water for cell recovery
Will do.

[0085]

Embodiment 12Utilization of various carbon and nitrogen sources  Transfer 50 ml of M5 medium in a 250 ml Erlenmeyer flask to agar.
Schizochytrium sp. S31 (AT
CC number 20888) or Troost thorium sp.
One colony of U42-2 (ATCC number 20891)
Inoculated. M5 medium is 2ml vitamin mix
And described in Example 4 except for 2 ml of the antibiotic mix.
Is the same as The culture is placed on a rotary shaker (200
rpm) at 30 ° C. Two days later, the culture
It had a medium density and was actively growing. This culture
With one of the following instead of glucose (at 5 g / l)
Of: dextrin, sorbitol, fructose, lacquer
Toast, maltose, sucrose, cornstarch, small
Wheat starch, potato starch, ground corn; or
Is one of the following (in 5 g / l) instead of glutamate
A: Gerizate, Peptone, Tripton, Casein, Ko
Immersion liquid, urea, nitrate, ammonium, whey
Or corn gluten meal
It was used to inoculate the flask containing the group. Times the culture
Ink for 48 hours on a shaker (200 rpm, 27 ° C)
Was added. Relative, showing growth on various organic substrates
Typical culture concentrations are shown in Tables 7-8.

[0086]

[Table 13]

[0087]

[Table 14]

[0088]

Embodiment 13Increases ω-3 HUFA content in shrimp
Of troost titride-based bait supplements for food  Thraustochytrium sp. 12B (ATCC No. 208
90) of the cellular biomass at 25 ° C. in M-5 medium
(See Example 6), produced in shake flasks. G
Roast Titrium sp.S31 (ATCC No. 2088)
8) Cellular biomass at 27 ° C in M-5 / 10 culture medium
Medium (see Example 9), produced in shake flasks.
Cells of each line were harvested by centrifugation. Pellets
Wash with single distilled water, then centrifuge again to remove 50% solids
Manufactured. Resuspend the obtained paste in seawater
After that, as a feed supplement,
Gave. This saltwater shrimp was previously an agricultural waste product
Bred and, as a result, its ω-3 HUFA content
Is very low, only 1.3-2.3% of the total fatty acids
(On average, saltwater shrimp caught in nature
6-8% ω-3 HUFA). This saltwater shrimp
(2 to 3 / ml) 1 liter beaker filled with seawater
To mix the aeration and the feed
Air stone was used. After adding food supplements, salt water
Shrimp samples are harvested periodically, washed and then
Fatty acid content is determined by gas chromatography.
Was. The results are shown in FIGS. Troasted chicken tribe
When the bait supplement is given as the finishing bait, the saltwater shrimp ω
-3 content within 5 hours when strain 12B is given,
Wild strain water within 11 hours
It can be increased to the same as the content of bi. Saltwater shrimp
Ω-3HUFA content of these dietary supplements for 24 hours
Up to far beyond the content of the wild
Can be In addition, these food supplements are
In the saltwater shrimp caught in
Significantly increases the reported DHA content.

[0089]

Embodiment 14Egg laying egg which is rich in ω-3HUFA
Troost Titolide-based feeding aid for litter
Feeding things  Thraustochytrium sp.S31 (ATCC No. 208
88) M-5 / 10 culture of the cellular biomass at 27 ° C
In a 10 liter fermentor (see Example 9)
did. Thraustochytrium sp.S31 (ATCC number
20888) cells are harvested by centrifugation and once with distilled water.
Wash and re-centrifuge to produce 50% solids paste
Was. The cell paste is then used in one of two ways:
Treated with: 1) frozen; or 2) ground corn
To produce a 70% solids paste,
Extrude at ~ 120 ° C and air dry. The resulting dried product is
Next, crush, analyze ω-3 HUFA content, and spawn
400 mg of ω-3 HUFA per day
(400 mg of ω-3HUFA)
/ 100 g feed). Laid eggs for about 45 days
Ω-3HU by sampling and gas chromatography
The FA was analyzed. 200-425mg ω-3HUF
A / Egg chicken is fed with ω-3 supplement DE
The eggs were laid (concentration normalized to 5000 mg fatty acid eggs)
Every time). When cooked, these eggs have no fishy odor
Was. The control hen is about 20mg ω-3HUFA
/ Eggs including eggs were laid. Control group and ω-3 assistance
Significant in the number of eggs laid by feeding hens
There was no significant difference. Obtained with feed supplements and control foods
There was no change in the yellow color of the egg.

[0090]

Embodiment 15Production of high-purity products (> 90% pure ω-3
HUFA or> 90% pure HUFA fatty acid mixture
object)  Thraustochytrium sp.S31 (ATCC No. 208
88) Cellular biomass in a 10 liter fermenter
Produced at 27 ° C in M-5 / 10 medium (see Example 9)
did. Cells of this strain were harvested by centrifugation. About 5
g cell paste, fill half with 0.5mm glass beads
350ml stainless steel from a bead beater bead mill
Placed in a less steel grinding chamber. The rest of that volume
Is filled with reagent grade MeOH and cells are washed twice for 3 minutes
While homogenizing. During the bead mill operation, stainless steel
The teal chamber was kept cool with an attached ice bath.

A solution of the crushed cells was poured into a flask, and chloroform and a 2M aqueous solution of NaCl were added thereto to make a final solution of about 1: 1: 0.9 (chloroform: MeOH:
Water). The solution was then poured into a separatory funnel and shaken several times to facilitate the transfer of lipids to the chloroform layer. After the solution was separated within a few minutes, the chloroform layer was collected in a flask, fresh chloroform was added to a separating funnel, and the extraction was repeated. Thereafter, the chloroform layer was extracted from the separating funnel, and the two chloroform layers were combined. The chloroform was then removed (and recovered) using a rotary vacuum evaporator operating at 40 ° C.
A part (300 mg) of the remaining lipid was taken out, and 50 ml was placed in a 150 mg Teflon (registered trademark) -lined bottle with a screw cap.
Of methanolic NaOH (10 ml of 0.3N NaOH diluted to 100 ml with methanol) at 60 ° C. for 6 hours. Then, non-hydrolyzable substances (steroids, hydrocarbons, etc.) are added to the separation funnel for 50m.
The layers were separated twice with 1 petroleum ether and the ether layer was removed by discarding in each case. The remaining solution was then acidified by adding 3 ml of 6N HCl and the free fatty acids were extracted twice with 50 ml of petroleum ether. Five ml of an ether solution containing free fatty acids was placed in three 13 mm × 100 mm test tubes, and the ether was removed by blowing the solution under a stream of nitrogen. Thereafter, 2 ml of ether, hexane or acetone was added to one tube, capped and placed in a dry ice-ethanol solution (-72 to -74 ° C), and the non-HUF
The A fatty acid crystallized. When the crystallization was considered complete, the culture tubes were placed in 50 ml polycarbonate centrifuge tubes filled with fine powdered dry ice.
The tubes were then placed in a centrifuge cooled to -10 ° C and centrifuged at 10,000 rpm for 3-5 minutes. Thereafter, the supernatant was quickly removed from each tube with a Pasteur pipette and placed in a clean culture tube. The solvent was removed from the supernatant by blowing nitrogen. The fatty acids were then placed in a tube with a Teflon-lined screw cap under nitrogen in methanolic H ₂ SO _4.
100 ° C. in (96 ml of MeOH, 4 ml of H ₂ SO ₄ )
For 1 hour. The fatty acid methyl ester was then subjected to gas chromatography (HP5890 gas chromatograph, Spelco SP2330 column; column temperature = 20).
0 ° C .; detector and injection part temperature = 250 ° C .; carrier gas = nitrogen). The composition of the resulting fatty acid mixture is 93.1% (ether) HUFA-23.4% C
22: 5n-6 + 69.7% 22: 6n-3; (hexane) 91.5% HUFA-66.8% 22: 6n-3
+ 22.1% 22: 5n-6 + 2.6% 20: 5n-
3; (acetone) 90.0% HUFA-65.6% 2
2: 6n-3 + 21.8n-6 + 2.6% 20: 5n-
3.

A mixture of fatty acids containing> 90% ω-3 HUFA can be obtained by performing the above-mentioned method on the harvested biomass of a strain of Troost titride, such as 12B (ATCC No. 20890).

[0093]General remarks  The following novel strains isolated according to the method of the present invention
Examples of the organisms disclosed and claimed in
can Type Culture Collection (ATCC), Melilla
Deposited in Rockville, Rand. Strain ATCC No. Deposit date Schizochytrium S31 20888 8/8/88 Schizochytrium S8 20889 8/8/88 Schizochytrium 12B 20890 8/8/88 Thraustochytrium U42-2 20891 8/8/88 Schizochytrium 23B 2089288/88/88

Although the present invention has been disclosed for a particular microbial strain, it is intended to cover all methods and strains obtained or usable in accordance with the teachings disclosed herein, which are well known to those skilled in the art. Such substitutions, modifications, and optimizations are all included in the present invention.

[Brief description of the drawings]

FIG. 1. Troost Titrium sp.UT42-2 (A
Figure 4 is a bar graph showing the effect of various media supplements on fatty acid yield using TCC number 20891) as a test strain. The above strains were isolated according to the selection method of the present invention.
The experimental method is described in Example 2. Vertical axis: F without supplement
Fatty acid yield based on FM culture medium control. Horizontal axis: 1) 2x "B"-vitamin mix, 2) 2
x "A" vitamin mix, 3) 2xPI metals, 4)
28 mg / l KH ₂ PO ₄ , 5) Combination treatment of 2) and 3) and 4), and 6) Specific addition of 480 mg / l KH ₂ PO ₄ .

FIG. 2 is a graph showing the amount of polyunsaturated fatty acid production, where □ indicates a strain newly isolated in the present invention, and ★ indicates a previously isolated strain. Each point indicates a certain strain, and the position of each point is determined by the weight% (horizontal axis) of the total fatty acids of ω-3 polyunsaturated fatty acids and the weight% (vertical axis) of all fatty acids which are ω-6 fatty acids. Is done. Only strains of the present invention have less than 10.6% (w / w) of total fatty acids at ω-6
And plotted at the point where at least 67% of the total fatty acids were at ω-3. Data from Table 4.

FIG. 3 is a graph showing the amount of polyunsaturated fatty acid production, where □ indicates a strain newly isolated in the present invention, and ★ indicates a previously isolated strain. Each point represents a certain strain, and the position of each point is the total fatty acid weight% (horizontal axis) of the total fatty acids of ω-3 polyunsaturated fatty acids and eicosapentaenoic acid (EPA C20: 5w3) (vertical axis). Is determined from the weight% of Only the strain of the present invention has 6
More than 7% (w / w) is ω-3, 7.8% of total fatty acids
(W / w) is plotted at the point where C20: 5w3.

FIG. 4 is a graph showing the composition of ω-3 polyunsaturated fatty acids, in which □ indicates a strain newly isolated in the present invention, and ★ indicates a previously isolated strain. Each point represents a separate strain. The value on the horizontal axis is the weight ratio of total ω-3 polyunsaturated fatty acids of C20: 5w3, and the value on the vertical axis is the weight ratio of total ω-3 polyunsaturated fatty acids of C22: 6w3. . Only the strains of the present invention were plotted at points showing either a weight ratio of C20: 5w3 of 28% or more, or a weight ratio of C22: 6ω3 of 93.6% or more.

FIG. 5 is a graph showing the growth of various newly isolated strains of the present invention and previously isolated strains at 25 ° C. and 30 ° C. The growth rate is shown normalized to the growth rate of the U-30 strain at 25 ° C. The previously isolated strain is indicated by its ATCC accession number. Table 5 shows the numerical data on the number of cell doublings per day.

FIG. 6 is a graph of total production in cells after induction by nitrogen limitation. The ash-free dry weight, total fatty acids and ω-3 polyunsaturated fatty acids were each plotted, as shown, normalized to the corresponding values of the 28211 strain. All strains are identified by ATCC accession numbers.

FIG. 7 is a graph of the fatty acid yield after growth in culture media containing the salt concentration shown on the horizontal axis. The indicated strain is a newly isolated S31 strain (ATCC No. 20888) (■)
And U42-2 strain (ATCC 20891) (+), and ATCC No. 28211, a strain that had been isolated in all cases.
(*) And ATCC No. 28209 (□). Fatty acid yields were determined for the S31 strain (AT
Based on the average growth rate indicated by CC No. 20888), the relative yield is plotted with the appropriate value being 1.00.

FIG. 8 is a graph showing an increase in the ω-3 polyunsaturated fatty acid content of total lipids in a saltwater shrimp, Artemia salina, bred with a troost titride strain (ATCC No. 20890) isolated by the method of Example 1. It is. EPA = C20:
5w3; DHA = C22: 6w3.

FIG. 9: Troost chicken isolated by the method of Example 1
Salted shrimp bred in the strain (ATCC No. 20888),
Ω-3 highly unsaturated fatty acids of all lipids in Artemia salina
It is a graph which shows an increase in content. EPA = C20: 5w
3: DHA = C2 2: 6w3.Literature  Ainsworth, G.C. (1973) "Introduction and keys to
the higher taxa. "In:The Fungi. An Advanced Treat
ise. Vol. 4B, G.C.Ainsworthet al. (Eds.), Academi
c Press, New York, pp. 107. Bahnweg, G. & Jackle, I. (1986) "A new approach t
o taxonomy of theThraustochytriales and Lybrinthul
ales. "In:The Biology ofMarine Fungi, S.T.Moss
 (ed.), Cambridge University Press, London, pp. 131
-140. Barr, J.S. (1981) "The phylogenetic and taxonomic
 implications offlagellar rootlet morphology among
 zoosporic fungi. "BioSystems14: 359-370. Barr, J.S. (1983) "The zoosporic grouping of plan
t pathogens. "In:Zoosporic Plant Pathogens: a mo
dern perspective, S.T.Buczacki (ed.), Academic Pre
ss, pp. 43-83. Bartnicki-Garcia, S. (1988) "THe cell wall: a cru
cial structure infungal evolution. "In:Evolutio
nary Biologyof the Fungi, A.D.M.Rayneret al. (ed
s.), Cambrindge University Press, pp. 389-403. CalBiochem Co. (1987).Biochemical / Immunochemical
Catalog. BehringDiagnostics, La Jolla, Californi
a. Cavalier-Smith, T. (1975) "The origin of nuclei a
nd of eukaryoticcells. "Nature256: 463-468. Cavalier-Smith, T. (1983) "A 6-kingdom classifica
tion and a unifiedphylogeny. "In:Endocytobiolog
y II: Intracellular Space asOligogenetic System,
H.E.A.Schenk and W. Schwemmler (eds.), De Gruyter
(Berlin), pp. 1207-1034. Chamberlain, A.H. and Moss, S.T. (1988) "The thra
ustochytrids: a protist group with mixed affinitie
s. "BioSystemstwenty one: 341-349. Cerda-Olmeda, E. & Lipson, E. (1987)Phycomyces,
Cold Springs Harbor Laboratory, Cold Springs Harbo
r, New York. Dick, M.W. (1973) "Saprolegniales." In: The Fun
gi. An AdvancedTreatise, Vol. 4B, G.C. Ainsworthet
 al(eds.), Academic Press, New York, pp. 113-144. Ellenbogen, B.B. et al. (1969) "Polyunsaturated f
atty acids of aquaticfungi: possible phylogenetic
 significance. "Comp. Biochem.Physiol.29: 805-81
1. Emerson, R. (1950) "Current trends of experimenta
l reserch in theaquatic Phycomycetes. "Ann. Rev.M
icro.Four: 169-200. Erwin, J. (1973) "Comparative biochemistry of fat
ty acids ineukaryotic microorganisms. "In:Lipid
s and Biomembranes of Eukarytic Microorganisms, J.
Erwin (ed.), Academic Press, New York, pp. 41-143. Findlay, R.H.et al. (1986) "Biochemical indicato
rs of the role offungi and Thraustrochytrids in ma
ngrove detrital systems. "In:The Biology of Marin
e Fungi, S.T.Moss (ed.), CambridgeUniversity Pres
s, London, pp. 91-103. Fuller, M.S.et al(1964) "Isolation and pure cu
lture of marinePhycomycetes. "Mycologia56: 745-7
56. Gellerman, J.L. & Schlenk, H. (1979) "Methyl-dire
cted desaturation ofarachidonic to eicosapentaenoi
c acid in the fungus,Saprolegniaparasitica. "Bio
chem. Biophys. Acta573: 23-30. Goldstein, S. (1963) "Development and nutrition o
f new species ofThraustochytrium. "Am. J. Bot.Five
0: 271-279. Goldstein, S.et al. (1969) "Biology of a problem
atic marine fungus,Dermocytidium sp. II. Nutritio
n and repiration. "Mycologia61: 468-72. Hiri, H.et al. (1980) Nucl. Acids Res. 8: 5535 55
39. Hunter, J.E. (1987) "Fish oil and other omega-3 s
ources. "J. Am. OilChem. Soc.64: 1592-1596. Kates, M. (1986)Techniques of Lipidology: Isola
tion, Analysis and Identification of Lipids, Elsevi
er, Amsterdam.Kazama, F. (1980) "The zoospore of Schizoshytrium
 aggregatum. Can J. Bot.58: 2434-2446. Kyle, D.J. (1987) "Microalgae as a source of EPA-
containing oils. "Abstract of talk presented at Wor
ld Conference for Fats & Oils, Hamburg, West German
y. J. Am. Oil Chem. Soc.64: 1251. Leedale, G. (1974) "Howmany are the kingdoms of o
rganisms. "Taxontwenty three: 261-270. Lepage, G. and Roy, C. (1984) "Improved recovery
of fatty acidthrough direct transesterification wi
thout prior extraction orpurification. "J. Lipid
Res.twenty five: 1391-1396.Margulis, L. (1970) Origin of Eukaryotic Cells, Y
ale University Press, New Haven.Margulis, L. and Sagan, D. (1985) "Order amidst a
nimalcules: theProctoctista kingdom and its undul
ipodiated cells. "BioSystems18: 141-147. Mannella, C.A.et al. (1987) "Interrelatedness of
 5S RNA sequences invested by correspondence an
alysis. "J. Mol. Evol.twenty four: 228-235. Moss, S. (1986) "Biology and phylogeny of the Lab.
rinthulales andThraustochytriales. "In:The Biol
ogy of Marine Fungi, S.T.Moss (ed.), Cambridge Uni
versity Press, London, pp. 105-130.Perkins, F.O. (1976) "Fine structure of lower mar
ine and estuarinefungi. "In:Recent Advances in
marine Mycology, E.B.GarethJones (ed.), Elek Scie
nce, pp. 279-312.Pigot, G.M. (1989), "The need to improve omega-3
content of culturedfish, "World Aquaculture20: 63
-68. Perkins, F. (1974) "Phylogenetic considerations o
f the problematicthraustochytriaceous-labrinthulid
-Dermocystidium complex based onobservations of fi
ne structure. "Veroff. Inst. Meeresforsch.Bremer
h. Suppl.Five: 45-63. Pohl, P. and Zurheide, F. (1979) "Fatty acids and
 lipids of marinealgae and the control of their bi
osynthesis by environmentalfactors. "In:Marine
Algae in Pharmaceutical Science, H. Hoppeet al(e
ds.), W. de Gruyter, Berlin, pp. 473-524. Poyton, R. (1970) "The charaterization ofHyalloc
hlorella marina gen.et sp.nov.a new colorless co
unterpart of Chlorella. "J. Gen. Microbiol.62: 17
1-188. Ryther, J.H. (1983) "Cultivation of macroscopic m
arine algae. "In:Solar Energy Research Institute
Aguayic Species Program Review.Proc. Of the March
1983 Principal Investigators Meeting.SERI / CP-231-1
946, pp. 79-88. Sargent, J.et al. (1989), "The lipids," inFish
Nutrition, SecondEdition, J. Halver (ed.), Academi
c Press, pp. 153-218.Schlenk, H. (1954) "Urea inclusion compounds of f
atty acids. "Prog.Chem. Fats and other LipidsTwo:
243: 267. Schneider, J. (1976) "Cultivation of Microorgnism
s. Section 3.2: Fungi. "In:Marine Ecology, Vol.
3, Part 1. Cultivation, O. Kinne (ed.), Wiley Inte
rscience. Sigma Chemical Co. (1988). Catalog: Biochemical
and Organic Compounds for Reserch.Sigma Chemical C
o., St. Louis, Missouri. Simopoulos, A.P. et al. (1986)Health Effects of
Polyunsaturated FattyAcids in Seafoods, Academic P
ress, New York. Sorokin, C. (1973) "Growth measurements: dry wei
ght packed cellvolume, and optical density. "In:
 Handbook of PhycologicalMethods: Culture Methods
and Growth Measurements, J.R. Stein (ed.), Cambrid
ge University Press, Cambridge, pp. 321-343.Sparrow, F.K. (1960)Aquatic Phycomycetes. Unive
rsity of MichiganPress, Ann Arbor, pp. 37-38. Wassef, M. (1977) "Fungal lipids." Adv. Lioid Re
s.Fifteen: 159-232. Weete, J.D. (1980)Lipid Biochemistry of Fungi an
d Other Organisms.Plenum Press, New York. Yamada, H.et al. (1987) "Production of arachidon
ic acid andeicosapentaenoic acid by microorganism
s. "Abstract of talkpresented at Wold Conference
for Fats & Oils, Hamburg, WestGermany. J. Am Oil
Chem. Soc.64: 1254.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 1/12 C12N 1/12 A B C 1/14 1/14 A B C // (C12P 7/64 (C12P 7/64 C12R 1:89) C12R 1:89) (C12P 7/64 (C12P 7/64 C12R 1: 645) C12R 1: 645)

Claims (6)

[Claims] 1. An organic carbon source, assimilable nitrogen, a salinity below seawater salinity, and at least about 15
Cultivating a microorganism selected from microorganisms of the order Thraustochytriales, excluding Thraustochytrium and Schizochytrium, and capable of efficiently producing ω-3 polyunsaturated fatty acids, in a culture medium having a temperature of 0 ° C. Method for producing ω-3 polyunsaturated fatty acid. 2. The method of claim 2, further comprising selecting from ω-3 polyunsaturated fatty acids or microorganisms of the order Thraustochytriale excluding the genera Thraustochytrium and Schizochytrium;
3 During post-harvest processing of microorganisms capable of producing polyunsaturated fatty acids efficiently, BHT, BHA, TBHQ, ethoxycuin, β-carotene, vitamin E and vitamin C
A compound selected from the group consisting of: ω-3 polyunsaturated fatty acids or microorganisms of the order Thraustochytriale except genus Thraustochytrium and Schizochytrium, which efficiently produces ω-3 polyunsaturated fatty acids. 2. The method of claim 1, comprising adding to the resulting microorganism. Further comprising extracting lipids from a microorganism selected from the microorganisms of the order Thraustochytriales excluding the genera Trous thythotrium and Schizochytrium and capable of efficiently producing ω-3 highly unsaturated fatty acids; A method for producing an ω-3 polyunsaturated fatty acid according to claim 1. 4. In the above extraction, a) disrupting microbial cells to obtain disrupted cells; b) solvent extracting the lipid mixture from the disrupted cells; c) hydrolyzing the lipid mixture; 4. The method of claim 3, comprising cooling and crystallizing the non-highly unsaturated fatty acids in the mixture. 5. Schizochytrium of ATCC No. 20888, Schizochytrium of ATCC No. 20889, ATC
Thraustochytrium with C number 20890, Thraustochytrium with ATCC number 20891, ATCC number 2
0892, which is a mutant strain belonging to the order Thraustochytriales other than the genera Thraustochytrium and Schizochytrium, which is capable of producing ω-3 highly unsaturated fatty acids. Can grow over a wide temperature range, especially between about 15 ° C. and 48 ° C., and have a wide range of salinity levels, for example,
A unicellular microorganism that is capable of growing at low salinity levels, such as a salinity level that results in a conductivity between, and is selected from the group consisting of non-filamentous microorganisms. 6. a) (i) a microorganism which is derived from Schizochytrium of ATCC No. 20888 and is a mutant strain belonging to the genus Thraustochytrium and the genus Thraustochytriale excluding the genus Schizochytrium, wherein the strain is ω-3 highly unsaturated. It can produce fatty acids and has a wide temperature range, especially about 15
Microorganisms capable of growing between 0 ° C. and 48 ° C., capable of growing at low salinity levels, such as salinity levels resulting in a wide range of salinity, for example, between 5 and 40 mmho / cm, and not being filamentous; ii) a microorganism derived from Schizochytrium of ATCC No. 20889, which is a mutant strain belonging to the order Thraustochytriale except genus Thraustochytrium and Schizochytrium, which is capable of producing ω-3 highly unsaturated fatty acids; It can grow over a wide temperature range, especially between about 15 ° C. and 48 ° C., and has a wide range of salinity levels, especially for example 5-40 mmho.
microorganisms that can grow at low salinity levels, such as salinity levels that result in a conductivity of between 0.5 / cm and non-filamentous; and (iii) derived from ATCC No. 20890 Thraustochytrium, Thraustochytrium and Schizochytrium. A microorganism which is a mutant strain belonging to the order Thraustochytriales except for the genus, which can produce ω-3 polyunsaturated fatty acids, and which can grow in a wide temperature range, particularly between about 15 ° C. to 48 ° C.,
Microorganisms that can grow at low salinity levels, such as those that provide a wide range of salinity, especially conductivity between 5 and 40 mmho / cm, and are not filamentous; (iv) from ATCC no. A derived microorganism, which is a mutant strain belonging to the order Thraustochytriale excluding the genera Thraustochytrium and Schizochytrium, which is capable of producing ω-3 polyunsaturated fatty acids and has a wide temperature range, particularly about 15 ° C. ° C to 48 ° C
Microorganisms that are capable of growing between a wide range of salinity levels, especially low salinity levels such as those that provide a conductivity of, for example, between 5 and 40 mmho / cm, and are not filamentous; (v) ATCC numbers A microorganism belonging to the order of the Thraustochytriales except Thraustochytrium and Schizochytrium, which is derived from the Tauostitolium of 20892, and is capable of producing ω-3 polyunsaturated fatty acids. Temperature range, especially about 15 ° C to 48 ° C
Microorganisms that can grow between a wide range of salinity levels, especially low salinity levels such as those that provide a conductivity of, for example, between 5 and 40 mmho / cm, and are not filamentous. An edible product comprising microorganisms to be isolated, or lipids extracted therefrom; and b) edible substances.